I think the initial seeding you gave may be very less to proliferate. You can increase the cell number and see the proliferation.

Dear Mamatha M Pillai,

Many thanks for your answer. I am culturing 10000 cells per a well of 96 well plate, is this seeding density low?

hESC don't do well in single cells, typically. Normally, they are treated with a Rho/Rock inhibitor Y27632 for 24 hours. The Rho/Rock pathway helps cells sense their environment. mESC are typically much easier to culture than hESC. I never worked with them, but it could help your situation.

The cell number sounds enough. What's your hydrogel concentration? Too viscous gel might be an issue.

Dear Dr. Fayyazzadeh,

Many thanks for your answer.

I am trying to find the optimum concentration for my hydrogel;therefore, I have started from 10% to 50% hydrogel, but the cell viability is low for all samples. Should I start from even lower concentrations?

Regards,

Yes, it sounds way too much. I would have started with 5% and adjusted the concentration thereafter. Make sure to use a control as well with no gel in your culture media.

Dear Dr. Fayyazzadeh,

Isn't 5% too low? then why are we adding hydrogel?

I would suggest starting with the lowest possible concentration and then adjust it, especially when cellular viability is an issue.

Dear friends many thanks for your precious ideas. I raised my seeding density to 100000 cell/well according to some articles ,it worked well but the problem is that after removing cell containing hydrogels from each well,there are still too many cells adherent to the bottom of well. I am trying to trypsinize and detach them from well inorder to count them but they are too adherent. what should I do? even after 4min trypsinizing in 200 microliter trypsin I can't detach them from surface of 96 well plate.

you can use my hydrogel system for that